1. Field of the Invention
The present invention relates to mutants of streptokinase and their covalently modified forms. The present invention utilizes the homogenous, site-specific and defined PEG modification of streptokinase and its related variants with substitutions, additions, deletions or domain fusion constructs to allow their usage in the form of improved protein therapeutics.
2. Background of the Invention
Thrombus (blood clot) development in the circulatory system can cause vascular blockage leading to fatal conditions. Development of clot and its dissolution is a highly controlled process for the hemostasis. Any deviation from a normal hemostasis leads to various clinical conditions such as stroke, pulmonary embolism, deep vein thrombosis and acute myocardial infarction. Patho-physiological conditions emerging out of failed hemostasis needs immediate clinical attention. The most practiced medical intervention for such cases is intravenous administration of thrombolytic agents (Collen et al., 1988; Collen, 1990; Francis and Marder, 1991). The most commonly used thrombolytic agents include Streptokinase (SK), Urokinase (UK) and the tissue type plasminogen activator (tPA). Numerous pharmacoeconomic appraisal of use of different thrombolytics in the management of acute myocardial infarction have been carried out in the past (Mucklow, 1995; Gillis and Goa, 1996). Banerjee et. al., 2004, have reviewed the clinical usefulness of streptokinase and its applicability as a drug of choice. As far as clinical efficacy is concerned both streptokinase and tPA fare equally well but due to several fold low cost and a slightly better in vivo half life, streptokinase is the most preferred thrombolytic worldwide (Sherry and Marder, 1991, Wu et al., 1998). Also, the use of tPA is slightly more likely to cause strokes, the major side effect for both the drugs. However streptokinase, being a bacterial protein is antigenic in nature and may give rise to clinical complications such as allergic response or hemorrhage. Also, the circulating half-life (15-30 min) of streptokinase is not sufficient for effective thrombolysis (Wu et al., 1998).
Despite all these, in recent years, thrombolytic therapy with fibrinolytic agents, such as Streptokinase (SK), tissue plasminogen activator (TPA) or urokinase (UK) has revolutionized the clinical management of diverse circulatory diseases e.g., deep-vein thrombosis, pulmonary embolism and myocardial infarction. These agents exert their fibrinolytic effects through activation of plasminogen (PG) in the circulation by cleavage of the scissile peptide bond between residues 561 and 562 in PG. As a result, inactive zymogen is transformed to its active form, the serine protease, plasmin (PN), which then circulates in the system and acts on fibrin to degrade the later into soluble degradation products. It may be mentioned here that PN, by itself, is incapable of activating PG to PN; this reaction is catalyzed by highly specific proteases like TPA, the SK-plasminogen complex, and UK, all of which possess an unusually narrow protein substrate preference, namely a propensity to cleave the scissile peptide bond in PG in a highly site-specific manner. However, unlike UK and TPA, SK has no proteolytic activity of its own, and it activates PG to PN “indirectly” i.e. by first forming a high-affinity equimolar complex with PG, known as the activator complex (reviewed by Castellino, F. J., 1981). The activator complex then acts as a protease that cleaves other, substrate molecules of PG to PN.
Regardless of tremendous advances in therapeutic use of streptokinase and other bacterial thrombolytics, there are several shortcomings that limit the usefulness of these polypeptide drugs. These disadvantages include their susceptibility to degradation by proteolytic enzymes, short circulating half-life, short shelf-life, rapid kidney clearance and their propensity to generate neutralizing antibodies. These shortcomings are also sometimes inherent to many other polypeptide drugs that are non human in origin. This aspect in general is reviewed by Roberts et. al; 2002. Various attempts were made to overcome these short comings in polypeptide drugs, such as altering the amino-acid sequences to reduce proteolysis or antigenicity, fusing the polypeptides to globulin or albumin domains to improve half-life etc. (Osborn et. al., 2002). These methods provided little help to the problem and came along with associated burden. The major breakthrough in this area was method of protein PEGylation that provided single solution to multiple problems. PEG (Poly Ethylene Glycol) is formed by polymerizing number of repeating subunits of ethylene glycol to give rise to linear or branched PEG polymers of tailored molecular masses. Once covalently conjugated with PEG the protein or polypeptide shows improved pharmacokinetic and pharmacodynamic properties such as increased water solubility, decreased renal clearance and often substantially limited immune reactivity (Moreadith et. al., 2003, Doherty et al., 2005, Basu et. al., 2006). The PEG conjugation also makes the molecule proteolytically less susceptible. The decreased receptor interaction or interaction with cell surface proteins that follows the PEG addition also helps to reduce adverse immunological effects. PEGylated drugs are also more stable over a wide range of pH and temperature changes (Monfardini et al. 1995). Use of PEG is FDA approved for therapeutics and it shows virtually no toxicity and eliminated from the body intact by either kidneys or in faeces. The beneficial features of PEG conjugation can be potentially imparted to SK to make it a more effective and safer thrombolytic. Attempt of SK PEGylation is reported in literature (Rajagopalan et. al., 1985) using a relatively non-specific chemical modification reaction. The therapeutic uses of such modifications were severely limited by highly compromised plasminogen activation ability. Also the nature of modification was poorly defined and heterogeneous in nature. The reason for this heterogeneity was the chemistry used for PEG modification that does not target modification of a specific site. This could be the reason why such modification strategy was not utilized for the development of improved SK based thrombolytics.
The term streptokinases used anywhere in the text collectively refers to: variants of streptokinase, any of its functional fragments, functional muteins, isolates from different species and fusion products obtained through attachment of oligo or polypeptides of natural or artificial origin.
It is known that different functional groups present in a protein can be utilized for PEG introduction. The most commonly employed techniques are derivatization of lysine residues or cysteine residues in the protein. Alpha-amino group at the N-terminus can also be exploited for single homogenous conjugation of PEG in proteins (Baker et. al., 2006). However, the use of cysteine residues to bear the incorporated PEG groups is particularly advantageous since, potentially, the —SH groups can be targeted in a site-specific mode particularly if the protein bears or made to bear a very limited number of cysteine residues. It is not an exaggeration to state that PEG conjugation becomes an art form when the protein is devoid of any cysteine since it leaves a virtual blank canvass for cysteine addition, insertion or substitution for site-specific PEG “painting”, or decoration, of proteins. Since potentially addition of cysteines into the cysteine free background can have adverse effects on the protein function. Therefore, the selection of sites for preparation of cysteine variants requires careful planning and execution. In contrast to, say, Lysine based modifications for PEGylation, although the chemistry is well defined, heterogeneity in reaction is a big disadvantage. In the case of SK, a large number of lysine residues are evenly spread all along the polypeptide and hence limit the possibility of homogenous site-specific PEG conjugation. More interestingly, there is no natural Cysteine present in the Streptokinase molecule (Malke et. al., 1985), thus making it possible to generate various Cysteine variants of streptokinase. Also there are no free cysteines in the natively folded covalent variants of SK derived by fusion with fibrin binding domains (ref. U.S. Pat. No. 7,163,817). This renders the possibility of making various free cysteine containing variants of Clot-specific streptokinase without actually interfering with the normal refolding of the cysteine-rich protein (all the cysteine residues being engaged in disulfide bond formation). The free Cysteine(s) introduced can be reacted with various thiol-reactive reagents including PEG to generate Cysteine adduct/s of these proteins.
Streptokinase (SK) is a generic name for a secretory protein produced by a variety of hemolytic streptococci that has the ability to induce lysis of plasma clots (Tillet and Garner, 1933). Because it can be easily and economically produced from its parent source, or through rDNA technology from suitable heterologous hosts, SK is very cost effective and thus is a major thrombolytic drug particularly for the cost-conscious markets world-wide. SK has been found very effective in the clinical treatment of acute myocardial infarction following coronary thrombosis (ISIS-3, 1992) and has served as a thrombolytic agent for more than three decades. However, it suffers from a number of drawbacks. It is known that the plasmin produced through the streptokinase mediated activation of plasminogen breaks down streptokinase soon after its injection (Rajagopalan et. al., 1985, Wu et. al., 1998). This limits the in vivo half-life of streptokinase to about 15 min (Wu et. al., 1998). Although streptokinase survives in circulation significantly longer than does another thrombolytic drug of choice, TPA (with a half-life less than 5 min; Ross, 1999; Ouriel, 2002), this is still short for efficient therapy (Wu et al., 1998). Because of the recognized shortcomings related to rapid in vivo clearance of the available plasminogen activators, attempts are underway to develop improved recombinant variants of these compounds (Nicolini et al., 1992, Adams et al., 1991, Lijnen et al., 1991; Marder, 1993, and Wu et al., 1998). Despite its inherent problems, streptokinase remains the drug of choice particularly in the developing countries because of its low relative cost (e.g., approximately US$ 50 or less per treatment compared to nearly US $ 1500 for TPA).
Streptokinase was first reported to cause lysis of blood clots by Tillet and Garner (1933). However, later it was established that the fibrinolytic activity of SK originates from its ability to activate human plasminogen (HPG, reviewed by Castellino, 1979). Streptokinase is mainly secreted by -hemolytic group A, C and G streptococci. SK is an activator of human PG though itself it is not a protease, rather it binds to human PG/PN and recruits other HPG molecules as substrate and converts these into product, PN. The latter circulates in the blood stream. Plasmin, being a non-specific protease, the generalized and immediate PN generation subsequent to SK injection results in large scale destruction of various blood factors leading to risk of hemorrhage, as also the dissolution of ECM and basement membrane (BM) and enhances bacterial invasiveness into secondary infection sites within the host body (Esmon and Mather, 1998; Lahteenmaki et al., 2001). Thus, there is an acute need to minimize the side-effects by designing improved SK analogs.
SK is currently being extensively used as a thrombolytic drug world wide since it is an efficient fibrin clot dissolver, yet it has its own limitations. SK being a protein produced from β hemolytic streptococci, its use in humans induces immunogenicity (McGrath and Patterson, 1984; McGrath et al., 1985; Schweitzer et al., 1991). The high titres of anti-SK immunoglobulins (Ig) generated after the first SK administration are known to persist in patients for several months to a few years (Lee et al., 1993). Thus, the anti-SK antibodies severely limit its use as future repeat therapy by either neutralizing SK upon administration (Spottal and Kaiser, 1974; Jalihal and Morris, 1990) or by causing a range of allergic reactions (McGrath and Patterson, 1984; McGrath et al., 1985).
As mentioned before, the use of streptokinase in thrombolytic therapy is hampered by the relatively short half-life (a few minutes) of this protein in vivo (which indeed is the case with all presently employed thrombolytic drugs), apart from its immunogenicity. It is observed that foreign proteins when introduced into the vertebrate circulation are often cleared rapidly by the kidneys. This situation becomes even more acute in case of streptokinase where progressively higher doses of the protein (to overcome antibody based rapid neutralization) can severely increase probability of allergic response/s, making the repeated administration essentially ineffective and dangerous. Attempts to solve these problems in general, are well documented in the literature where various physical and chemical alterations have been shown to be useful for generation of improved therapeutics, e.g. see: Mateo, C. et al 2000, Lyczak, J. B. & Morrison, S. L. 1994, Syed, S. et. al; 1997, Allen, T. M. 1997. The most promising of these to-date is the approach of modification of therapeutic proteins by covalent attachment of polyalkylene oxide polymers, particularly polyethylene glycols (PEG). PEG is a non-antigenic, inert polymer and is known to increase the circulating half-life of the proteins in the body (Abuchowski et al., 1984; Hershfield, 1987; Meyers et al., 1991). This allows the extended action of the drug in use. It is believed that PEG conjugation to proteins increases their overall size and hence reduces their rapid renal clearance. PEG attachment also makes the protein or polypeptide more water soluble and increases its stability under in vivo conditions along with markedly reducing immunogenicity and increasing in vivo stability (Katre et al., 1987; Katre, 1990). U.S. Pat. No. 4,179,337 discloses the use of PEG or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition.
Although the chemistry of PEG conjugation is mostly generic but strategic placement of PEG polymers in a therapeutic protein is of paramount importance to achieve successful outcomes. Availability of three dimensional structural information with functional hot spots earmarked through various solution studies, helps in designing mutational plan to keep the functionality intact.
The complete amino acid sequence of SK was determined by sequential Edman degradation analysis of SK fragments generated by cyanogen bromide and enzymatic methods (Jackson and Tang, 1982). The results established that the molecule of Mr 47,408 Da, contains 415 amino acids in a single polypeptide chain amino acid sequence.
The nucleotide sequence from S. equisimilis H46A (the prototype strain for SK production that is most often used therapeutically in humans) was sequenced by Malke and co-workers, in 1985. The transcriptional control of this gene has also been studied and the functional analysis of its complex promoter has been reported (Grafe et al., 1996). Considerable information exists, therefore, for effectively using this gene in producing streptokinase safely in relatively non-pathogenic microbes.